微管蛋白對神經突發生之研究

Abstract

形成一個有功能的神經系統仰賴於精密複雜的神經連結，而神經連結的產生則需要高度特化的細胞突出物稱為神經突 (neurite)。微管 (microtubule) 是神經細胞中最大量存在的細胞骨架，而微管也在神經突型態發生的每個步驟都扮演著不可或缺的角色。此研究計畫旨在研究微管細胞骨架在神經型態發生前期的功能。具體而言，我們將研究微管蛋白 (microtubule-associated protein) TPX2以及所有微管動力蛋白 (microtubule-based motors) 在神經突型態發生過程中所扮演的功能。 我們先前的研究顯示，抑制微管蛋白TPX2會影響胚胎癌細胞所分化成的神經細胞以及初代海馬迴神經細胞之神經突型態發生。另外，我們也發現TPX2位在神經細胞的中心體並與γ-tubulin有相同的位置。為更深入了解TPX2在神經突發生所扮演之功能，我們將研究抑制TPX2對微管細胞骨架之影響。首先，我們將觀察抑制TPX2對神經細胞中心體位置以及微管動態之影響。其次，我們會透過研究TPX2與γ-tubulin的交互作用來了解這兩種微管蛋白在中心體上的功能。此外，我們將透過大量表現TPX2來嘗試增強神經突生長以闡明此蛋白在神經突型態發生的功能。 第二，我們計畫以雷射鑷夾來捕捉並且移動中心粒，利用此方法研究中心粒在軸突起始過程中所扮演的功能。我們將使用CHO細胞來了解雷射鑷夾捕捉中心粒的可能性，並利用小鼠海馬迴神經元來研究改變中心粒位置對於神經型態發生所造成之影響。本研究將可首次釐清中心粒是否主動影響軸突起始。 第三，我們計畫利用shRNA，系統性地抑制所有的微管動力蛋白 (microtubule-based motors)，以研究神經突型態發生過程中每個微管動力蛋白的功能。我們將以高通量分析技術 (high-content assay) 紀錄、分析、歸類抑制每個動力蛋白所造成的神經型態改變。本研究將可初次一窺所有微管動力蛋白於神經突型態發生所扮演之角色。 總結我們三年期計畫的目標如下: 1. 研究TPX2於神經突型態發生所扮演之功能 (第一年) 2. 研究中心體於軸突起始所扮演之功能 (第二、三年) 3. 以高通量分析法研究微管動力蛋白於神經突型態發生所扮演之功能 (第二、三年)

A functional nervous system depends on the intricate network of neuronal connections. To form the neuronal connection, each neuron must establish highly specialized cellular protrusions called neurites. Microtubule is the most abundant cytoskeleton in the neurite and it plays indispensable roles in every steps of neurite morphogenesis. This proposal aims to elucidate the function of the microtubule cytoskeleton during early neurite morphogenesis. To be specific, this proposal intends to study the function of a microtubule-associated protein called TPX2 and every single microtubule-based motor in neurite morphogenesis. Our previous study showed that suppressing TPX2, a microtubule-associated protein, resulted in defective neurite morphogenesis in embryonic carcinoma cell-derived neurons and primary hippocampal neurons. In addition, we discovered that TPX2 localized to the centrosome and colocalized with γ-tubulin in neurons. To understand the function of TPX2 during neurite morphogenesis, we propose to examine the effect of suppressing TPX2 on centrosome positioning and microtubule plus-end dynamics. We also plan on examining the functional and physical interaction between TPX2 and γ-tubulin. Furthermore, we will explore the possibility of overexpressing TPX2 on enhancing neurite outgrowth. Second, we propose to verify the function of the centrosome in axon formation by physical repositioning using laser optical tweezers. The possibility of centrosome manipulation will be examined in CHO cells and the effect of centrosome repositioning will be examined in primary hippocampal neurons. This research will provide the first conclusive evidence on whether the centrosome is an active determinant in axon formation. Third, we propose to examine the function of all microtubule-based motors in neurite morphogenesis by systematically knocking down each and every motor using shRNA. An image-based high-content assay will be conducted and the phenotypic effect of each motor will be documented and classified. This research will provide the first comprehensive view of the function of all microtubule-based motors in neurite morphogenesis. The specific aims of our three-year project are as follow: 1. Characterizing the role of TPX2 in neurite morphogenesis (first year). 2. Examining the role of centrosomes in axon formation (second and third year). 3. Conducting high-content assay on motor-depleted neurons (second, and third year).